Capillary absorption spectrometer and process for isotopic analysis of small samples

ABSTRACT

A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional application of U.S. patent application Ser. No.13/757,460 filed 1 Feb. 2013, published as US Patent Publication No.:2014/0220700A1 on 7 Aug. 2014, now allowed.

STATEMENT REGARDING RIGHTS TO INVENTION MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under ContractDE-ACO5-76RL01830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to laser spectroscopy systemsand methods for detection of isotopologues. More particularly, thepresent invention relates to a laser ablation capillary absorptionspectrometer and process for determination of isotopologues in small(e.g., picomolar or sub-picomolar) samples.

BACKGROUND OF THE INVENTION

Isotope ratio (IR) mass spectrometry (MS) (i.e., IRMS), and laserablation (LA) mass spectrometry (MS) (i.e., LAMS) are conventionalapproaches for detecting isotopes. These systems typically involveconverting laser ablated material into carbon dioxide or carbonmonoxide, which species are then measured with a mass spectrometer toprovide an isotope ratio for analytes of interest. Tunable IR laserabsorption spectroscopies can measure raw isotopic differences for manydifferent gaseous samples at precisions better than 0.2/00. And, theycan offer a natural means to avoid isobaric interferences (¹⁴N¹⁴N¹⁶O,¹⁶O¹²C ¹⁶O, and etc.) because the IR absorption transitions are quitespecific to molecular structure. In fact, even pure isomers includingrearrangement isotopologues such as ¹⁴N¹⁵NO and ¹⁵N¹⁴NO, etc. can bereadily distinguished with tunable IR laser absorption spectroscopy.Laser-based isotope discriminators are also being developed to handle avariety of gaseous compounds since much less sample preparation isneeded to isolate isobaric interferences. And, laser-based systems andmethods can offer more flexibility in performing real-time or nearreal-time isotopic analyses of atmospheric gas samples in the field withdirect ingestion of mixed gas samples. However, most commercial laserabsorption-based devices are designed for fast in situ analyses ofambient gases. And, with long path-length absorption cells, such systemssuffer significant sample dilution. Further, while commercial IRMSsystems are available for measuring CO₂ isotopic differences,conventional optical cavity architectures currently require more than˜100 nanomoles of CO₂ to be effective. The technical literature reportsbest case minimum detectable absorbance (MDA) values forcontinuous-wave-QC laser systems used in concert with compact IR cavityring down spectroscopies of about 2.2×10⁻⁸. Furthermore, while IRabsorption in multi-pass or ring-down optical gas cells has been appliedto stable isotope measurements, only a fraction of a sample can beablated by the laser and released into the multi-pass or ring-downcavity in these systems. Given that analyses in these systems requireabout 200 to 300 nanomoles of sample, isotopic imaging at a 1 micronresolution is precluded. Secondary Ion Mass Spectrometry (SIMS) can alsoprovide data similar to those obtained with laser ablation, but at acost that is at least ten times greater and a throughput that is lower.SIMS also requires substantial sample preparation and must be performedunder ultra-high (e.g., 10⁻⁷ Torr or less) vacuum conditions, which havethe potential to alter the sample, and further precludes any in vivostudies. Accordingly new systems, devices, and processes are needed thatenable determination of stable isotopes at the single cell level orbetter. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention includes a new laser ablation capillary absorptionspectrometer (LA-CAS) and process that provide highly sensitive (i.e.,detection of molecular isotopologues at quantities down to 100femtomoles) and accurate (0.5‰ or better) stable measurements ofmolecular isotopologues in small samples containing down to 100femtomoles of material including samples containing biological materials(e.g., 1 to 10 cells).

The present invention includes an optical isolation device that couplesto an excitation laser and an absorption (or transmission) device.

The optical isolation device may include a turbulence generator thatinduces a random spatial and temporal variation in the index ofrefraction of a medium through which a light beam from the laser isdelivered as a function of time.

The medium may be a gaseous medium, a liquid medium, anaerosol-containing medium, a particulate-containing medium, andcombinations of these various media.

Turbulence generators may include, but are not limited to, e.g., heatsources, gas sources, forced gas sources, forced air sources, flowinggas sources, flame sources, sonic sources, liquid sources, forced liquidsources, flowing liquid sources, including combinations of these variousdevices.

The optical isolation device may include an aerosol or particulategenerator that delivers a distribution of aerosols and particulates ofselected sizes and velocities into a medium through which a light beamfrom the laser is delivered as a function of time. The distribution ofaerosols and particulates in the medium may induce a random spatial andtemporal variation in divergence angle and pointing angle of theexcitation beam when delivered through the medium. Aerosol andparticulate generators include, but are not limited to, e.g., aerosoldelivery devices, nebulizing devices, particulate delivery devices,including combinations of these various devices.

The optical isolation device may be positioned adjacent the excitationlaser in front of the absorption or transmission device.

The optical isolation device may include a heat source configured todeliver turbulent air at a selected “dither” (i.e., variable and random)frequency at one or more selected temperatures across the propagation(optical) path of the excitation beam delivered through a medium tooptically isolate the excitation laser from light reflected back to thelaser from an optically coupled absorption (or transmission) device.

The optical isolation device may be a sonic device configured to delivera random spread of sonic waves through a medium at a plurality oftemperatures above ambient across the propagation path of the excitationbeam emitted from the laser that provides a random spatial and temporalvariation in the index of refraction of the medium through which theexcitation beam is delivered en route to the absorption device and/orthe transmission device when directed through the spread of sonic waves.

The optical isolation device may deliver a turbulent gas or a turbulentliquid through a medium across the propagation path of the excitationbeam emitted from the laser that provides a random spatial variation inthe index of refraction of the medium through which the excitation beamis delivered through the turbulent gas or the turbulent liquid to theabsorption device or the transmission device.

The optical isolation device may deliver turbulent air at a temperatureselected in the range from about 150° C. to about 400° C.

The optical isolation device may include an enclosed liquid that iscoupled to a turbulence generator. The turbulence generator provides aturbulent liquid that provides a random spatial and temporal variationin the index of refraction of the light beam when delivered to theabsorption device or the transmission device through the turbulentliquid.

The optical isolation device may be a sonic device configured to delivera random spread of sonic waves in a medium at a plurality oftemperatures above ambient across the propagation path of the light beamfrom the laser that provides a random spatial and temporal variation inthe index of refraction of the medium when light from the laser isdirected through the spread of sonic waves in the medium.

One or more spatial filters may be positioned adjacent the opticalisolation device at a tilt angle that selects a subset of rays from theexcitation beam at a selected pointing angle and selected beam spreadinto the absorption or transmission device. The spatial filteringdevices may deliver the excitation beam off-axis to the absorption ortransmission device.

Spatial filtering devices include, but are not limited to, e.g., lenses,slits, pin holes, or combinations of these various devices.

The optical isolation device may randomize the spread of the excitationbeam wave fronts delivered through a medium to the absorption ortransmission device to eliminate feedback noise stemming fromreflections and/or scatter in the excitation beam returning back to theexcitation laser.

The medium through which the light beam is delivered may be a gaseousmedium, a liquid medium, an aerosol-containing medium, aparticulate-containing medium, and combinations thereof.

The optical isolation device can randomize the pointing angle and thespread of the light beam delivered to the absorption (or transmission)device to provide the random spatial variation as a function of timethat suppresses feedback noise induced by reflection of the light beamincident upon and/or from within the absorption or the transmissiondevice back to the laser device.

The optical isolation device can provide a signal-to-noise ratio for anabsorption sensitivity measurement obtained from the absorption deviceand/or a transmission sensitivity measurement obtained from thetransmission device that is enhanced by a factor of at least 10 timescompared to a system or device absent the optical isolation device.

The randomizing source may be an aerosol source configured to deliver arandom spread of aerosols with a random distribution of sizes andvelocities across the propagation path of the light beam when the lightbeam is directed through the spread of aerosols in the medium to theabsorption device or the transmission device.

The optical isolation device may be a vapor-nebulizing device configuredto deliver a random spread of aerosols of a selected size at a pluralityof angles and/or a plurality of temperatures above ambient across thepropagation path of the light beam that provides the random spatialvariation in the index of refraction of the light beam delivered to theabsorption device and/or the transmission device when directed throughthe spread of aerosols.

The optical isolation device may include an enclosed liquid maintainedat a random temperature above ambient that provides a random spatial andtemporal variation in the index of refraction of a medium through whichthe excitation beam is delivered en route to the absorption device orthe transmission device when directed through the liquid.

The medium may include an open volume or an enclosed volume.

The present invention also includes a capillary absorption spectrometer(CAS) that provides sensitive, accurate, and stable isotope measurementsin small samples including those containing biological materials down toa single cell level. The CAS spectrometer may include: an excitationlaser configured to supply a non-pulsed excitation beam to an absorptiondevice. The absorption device may include an absorption volumeconfigured to receive a sample gas containing analytes therein thatabsorb in the infra-red region when an excitation beam from anexcitation laser is introduced therein.

The spectrometer may include a (signal) modulation device configuredwith a second or higher order bias-T (diplexer) that couples to theexcitation laser. The bias-T may suppress harmonics from a repetitivelinear current ramp source and sub-harmonics from a dither-modulatedcurrent source in the modulation device such that the amplitude of theharmonics and the sub-harmonics is at least 60 dB lower than themodulation waveform selected and provided to the excitation laser.

The modulation device may deliver a frequency (f)-modulated excitationbeam to the absorption or transmission device, where (f) is ½ thein-quadrature condition of the residual amplitude modulation (RAM)distortion frequency (response) of a semiconductor laser.

The spectrometer may also include an optical isolation device that ispositioned between the excitation laser and an absorption device. Theoptical isolation device may randomize the pointing angle and the spreadof the excitation beam that suppresses feedback noise induced byreflection of the excitation beam incident upon, and from within, theabsorption device back to the excitation laser. The spectrometer mayalso include a detection device that couples to the absorption deviceand configured to detect absorption features for one or more analyteswhen present in the sample gas introduced in the absorption device.

The CAS spectrometer may provide a molar sensitivity of between about 10nanomoles and about 100 femtomoles for isotopic ratio measurements. TheCAS spectrometer may provide a precision for isotopic ratio measurementsof at least about 2‰ or better.

The CAS spectrometer may include a laser ablation device that is coupledto a catalytic reactor that is coupled to a transfer device. The laserablation device is configured to deliver ablated sample particulates tothe catalytic reactor for combustion therein. The catalytic reactor isconfigured to convert the sample particulates received from the ablationdevice into at least one molecular gas. The transfer device isconfigured to transfer the at least one molecular gas into the IRabsorption device for analysis therein.

The spectrometer may include a laser ablation device and a catalyticreactor coupled to the IR absorption device.

The method may include introducing a sample to the ablation device in acarrier gas. The carrier gas may include or be composed of, e.g., air,nitrogen, argon, helium, oxygen, carbon dioxide, and combinations ofthese various gases. The carrier gas may also be a CO₂-containing gas.

The catalytic reactor may be configured to oxidize or reduce a targetelement selected from C, N, O, S, P including combinations of theseelements present in the sample received from the laser ablation devicethat yields gas-phase molecular species in a form that can be probed byIR absorption. Gas-phase molecular species include species: 1) suitablefor IR absorption when introduced into the IR absorption device, or 2)that provide a sufficient IR absorption signal when introduced into theIR absorption device that permits determination of elemental ratios forisotopes therein.

The laser ablation device may be configured to ablate a small sampledefined by a quantity of material down to 100 femtomoles intoparticulates. The catalytic reactor may be configured to oxidize orreduce particulates received from the laser ablation device and combustthe particulates to convert same to one or more molecular gases. Theabsorption device may be configured to measure the absorption signal oftarget elements in the combusted gas sample received from the catalyticreactor and provide an isotopic ratio measurement of the target elementsin the sample with a precision of at least about 2‰ or better.

The transfer device may transfer analytes from the catalytic reactor ata first (high) pressure and deliver the analytes into the absorptiondevice at a 2^(nd) (lower) pressure without a loss of analytes.

The transfer device may be a conduction-limited transfer device thatprovides continuous transfer of analytes from the catalytic reactor atthe first (high) pressure to the IR absorption device at the second(lower) pressure in concert with a graded reduction in pressure.

The transfer device may be a cryogenic transfer device. The cryogenictransfer device may include a cryogenic trap. The cryogenic trap may beconfigured 1) to receive analytes from the catalytic reactor at thefirst (high) pressure and to simultaneously condense and trap theanalytes therein, 2) to isolate and evacuate the transfer device tomatch the second (lower) pressure of the absorption device, and 3) toevaporate and transfer the analytes into the absorption device at thesecond (lower) pressure. The cryogenic transfer device may be configuredto cycle the pressure between the first (high) pressure that matches theoutput pressure of the catalytic reactor while cryogenically trappingthe analyte in the cryogenic trap and the second (lower) pressure andevaporating the sample while simultaneously transferring the analyteinto the absorption device at the second lower pressure that matches theinput pressure of the IR absorption device.

The first pressure may be selected from about 100 Torr (0.13 atm) andabout 760 Torr (1 atm). The second (2^(nd)) pressure may be selectedfrom about 25 Torr (0.03 atm) or below. In some applications, the 2^(nd)pressure may be selected from about 1 Torr (0.001 atm) to about 25 Torr(0.03 atm).

The excitation laser may be a continuous-wave laser.

The CAS spectrometer may include one or more spatial selection orfiltering devices positioned to deliver the excitation beam from theexcitation laser off-axis to the absorption device through the medium ofthe optical isolation device to suppress reflections of light from theabsorption device back to the excitation laser.

The absorption device may be a capillary optical waveguide. Theabsorption device may be a tapered hollow wave guide with an internaldiameter that varies as a function of length, or an optical fiber withan internal diameter that is a fixed diameter. The absorption device maybe a hollow wave guide having dimensions of about 1-mm ID with aphysical path length of about 0.8 meter.

The absorption device may include an absorption volume at or below 10cm³. The absorption device may include an optical path length of about10 cm or greater.

The absorption device may be configured to contain a total gas pressureat or below 25 Torr (0.03 atm). The total gas pressure may be a combinedpressure for the sample gas plus the carrier (or buffer) gas.

The absorption device may be configured to receive a sample gascontaining one or more analytes therein.

The detection device may be configured to resolve 1f or 2f absorptionsignals of one or more analytes introduced in a sample gas to thecapillary absorption (or transmission) device.

The modulation device may include a dither-modulated current source thatdelivers a dither-modulated frequency. The dither-modulated frequencymay be tuned to a frequency that is one-half (½) the Residual AmplitudeModulation (RAM) distortion frequency (response) of the excitationlaser. Tuning refers to the process in which the frequency of a receivercircuit of a detector of may be continuously adjusted (e.g., over afrequency range being scanned) to locate a frequency where analytesabsorb and where the frequency of the absorption signal of the selectedanalyte(s) may be detected.

The modulation device may include both a repetitive linear current rampsource and a dither-modulated current source. The modulation device maybe configured to deliver frequencies that are non-integer fractions offundamental reference frequencies of these respective sources. Thenon-integer frequencies delivered by the modulation device may suppresscross talk arising from overlapping phase and/or frequency differencesbetween the repetitive linear current ramp source and thedither-modulated current source.

The repetitive linear current ramp source may deliver a frequency thatis at least a factor of 5 below the frequency delivered by thedither-modulated current source.

The detection device may include a phase-sensitive detection circuit.The phase-sensitive detection circuit may include a first circuitsegment having a Diplexer configured to deliver a detection signal fromthe absorption device as an input to a double-balanced mixing device,and a second circuit segment having a Band-Pass Filter (BPF) configuredto deliver a reference (drive) signal free of harmonic andintermodulation distortion from the dither-modulated current source ofthe modulation device as a separate input to the double-balanced mixingdevice. The double-balanced mixing device may deliver an output signalmade up of various combinations of the signals (i.e., mixed signals)received from the respective inputs.

The double-balanced mixing device (DBM) may couple to a 2^(nd) orgreater order Diplexer (bias-T) that is configured to 1) receive themixed output signal from the DBM as an input and 2) to isolate aselected absorption response signal for one or more selected analytespresent in the mixed signal.

The 2^(nd)-order or greater Diplexer of the phase-sensitive detectioncircuit may be configured to receive an output signal from the doublebalanced mixing device and to deliver a distortion free response signalas an output therefrom at a selected frequency.

The 2^(nd)-order or greater Diplexer may deliver the isolated responsesignal at 1-times (1f) or 2-times (2f) the dither-modulated frequency ofthe modulation device and that is 90 degrees out-of-phase with the RAMdistortion frequency of the excitation laser.

The 2nd-order or greater Diplexer may deliver a response signal with afrequency selected in the range from about 100 Hz to about 30 KHz withan isolation of about 30 dB per decade at the upper cutoff frequency.

The phase-sensitive detection circuit may suppress noise in theabsorption signal arising from the RAM distortion frequency receivedfrom the excitation laser and from harmonic distortion frequenciesreceived from the dither-modulated current source.

The present invention also includes a method for analyzing a sample.Samples may include low-volume of solid samples. The method may includeablating a picomolar quantity of a solid sample with a pulsed UVexcitation beam to convert the sample into sample particulates. Theablated sample particulates may be passed to a catalytic reactor. Theablated sample particulates may be combusted in the catalytic reactor toconvert the ablated sample particulates into a molecular gas. Themolecular gas from the catalytic reactor may be transferred through atransfer device at a first higher pressure into a capillary IRabsorption device at a second lower pressure.

The capillary IR absorption device for measurements of a quantity of atarget element less than 1 picomole may include a volume between about0.1 cm³ and about 1 cm³.

The sample in the absorption device may be analyzed with a capillaryabsorption spectrometer in concert with an IR absorption detector todetermine isotopic ratios of analytes in the sample. The sample may beanalyzed in the IR absorption device by probing with an IR laser beam.The analytes in the sample may be detected with a IR absorptiondetector.

The detector may be an infra-red sensitive detector. In someapplications, the detector is an indium-antimonide (InSb) detector.

The sample may be a biological isolate containing at least one singlecell. Analyses may be performed in vivo at ambient conditions inreal-time. The in vivo analyses may yield isotopic images of microbialsamples that allows tracking of nutrients in the microbial sample at thesingle cell level as a function of time.

Analyses may include a lower limit of detection for anisotope-containing sample gas down to a pressure of about 2 Torr (0.003atm).

The method may include calibrating the spectrometer with a solidcalibration standard. The calibration may be performed with a solidsample referenced to a known standard. The solid calibration standardmay include a polymer. The calibration may include introducing the solidisotopic or elemental standard into the laser ablation device to formparticulates that when introduced into the catalytic reactor formmolecular species that are the same as the analytes in the sample to bedetermined. The calibration may provide real-time, isotopic calibrationof the spectrometer that allows determination of the isotopic ratio ofanalytes in the sample. The calibration may include calibrating with anormalization method.

The purpose of the foregoing abstract is to enable the United StatesPatent and Trademark Office and the public generally, especially thescientists, engineers, and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the technical disclosure ofthe application. The abstract is neither intended to define theinvention of the application, which is measured by the claims, nor is itintended to be limiting as to the scope of the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating exemplary components of a capillaryabsorption spectrometer (CAS) of the present invention.

FIGS. 2a-2c show different generators used as components of an opticalisolation device, according to different embodiments of the presentinvention.

FIG. 3 shows devices that convert solid samples to molecular gasesanalyzed in the spectrometer of the present invention.

FIGS. 4a-4b show transfer devices of a cryogenic design used in concertwith the present invention.

FIGS. 5a-5b show transfer devices of a conduction limited design used inconcert with the present invention.

FIG. 6 shows schematics of an exemplary detection device coupled to amodulation device used in concert with the present invention.

FIG. 7 compares (2f) detection signals for representative isotopesobtained with and without optical isolation of the present invention.

DETAILED DESCRIPTION

A new laser ablation capillary absorption spectrometer (LA-CAS) andprocess are described that provide highly sensitive (down to 100femtomoles) and accurate (0.2‰ or better) stable isotope measurements ofmolecular isotopologues present in samples including samples containingbiological and other organic materials. The term “isotopologue” as usedherein refers to molecular species that differ only in their isotopiccomposition, i.e., the number of isotopic substitutions. Each member ofan isotopologue family has at least one atom with a different number ofneutrons than the parent molecule. Water, as an example, hasisotopologues that include: light water (HOH or H₂O); “semi-heavy water”with the deuterium isotope present in equal proportion to protium (HDOor ¹H²HO); “heavy water” with two deuterium isotopes of hydrogen permolecule (D₂O or ²H₂O); and “super-heavy water” or tritiated water (T₂Oor ³H₂O), where hydrogen atoms are replaced with tritium isotopes.Oxygen-related isotopologues of water include heavy-oxygen water (H₂¹⁸O) and the slightly lighter isotopologue containing the ¹⁷O isotope,(H₂ ¹⁷O). In the doubly labeled water isotopologue, both oxygen (O) andhydrogen (H) are replaced by isotopes (D₂ ¹⁸O). Further aspects of thepresent invention are detailed in a publication by Kelly et al. in[Review of Scientific Instruments 83, 023101 (2012)], which reference isincorporated herein in its entirety. The following description includesa best mode of at least one embodiment of the present invention. It willbe clear from this description that the invention is not limited tothese illustrated embodiments but that the invention also includes avariety of modifications and embodiments thereto. While the invention issusceptible of various modifications and alternative constructions, itshould be understood, that there is no intention to limit the inventionto the specific form disclosed, but, on the contrary, the invention isto cover all modifications, alternative constructions, and equivalentsfalling within the spirit and scope of the invention as defined in theclaims. Therefore the present description should be seen as illustrativeand not limiting.

LA-CAS Spectrometer

FIG. 1 is a schematic showing a laser ablation (LA) capillary absorptionspectrometer (LA-CAS) 100 of the present invention. LA-CAS spectrometer100 may include a modulation device 2 coupled to an excitation laser 4.Modulation device 2 may be configured to deliver a current-controlledwaveform to excitation laser 4. Excitation laser 4 may be configured tosupply a non-pulsed excitation beam 6 to an absorption (or transmission)device 30. The current-controlled waveform 6 may include: a DC offsetcurrent that establishes the initial frequency of beam 6 emitted fromlaser 4, a repetitive linear current ramp (i.e., repeated cycles of upand down ramping) that sets the range over which the frequency of laser4 is scanned that may be added to or superimposed atop adither-modulated current that serves to modulate the output frequency ofbeam 6 emitted from laser 4. The output frequency of beam 6 may bemodulated around a center value determined by the sum of the DC offsetcurrent and the linear ramped current as a function of time.“Dither-modulated” as used herein means a variation in a selectedfrequency that is on the order of the transition line width relative tothe total frequency range being scanned. The term “transition” as usedherein means a discrete absorption frequency characteristic of aselected molecular gas (or target isotopologue) defined by thedifference in energy levels being probed by the excitation laser. Theterms “dither” or “dithering” as used herein refer to a sinusoidalvariation in current added in the modulation device to a DC currentoffset and a linear ramp current resulting in a variation in laserfrequency that is smaller (10 times to 20 times lower) in amplitudecompared to a total range being scanned [e.g., ½ wavenumber (cm⁻¹)] andon a time scale that is faster (at least a factor of 5 greater) than thefrequency of the repetitive current ramp. For example, if the linearcurrent ramp is repetitively ramped (cycled) at 50 kHz, the ditherfrequency may be 250 kHz or greater.

Lasers

Continuous wave lasers 4 suitable for use may include, but are notlimited to, e.g., Quantum Cascade (QC) lasers, Distributed Feedback(DFB) lasers, Inductively Coupled (IC) lasers, External Cavity (EC) QClasers, diode lasers, including combinations of these various lasers.

Laser 4 may be contained within a Dewar vessel 8 that maintains a stabletemperature for laser 4. In some embodiments, laser 4 may be cooled,e.g., with liquid nitrogen, or with a thermo-electric cooler (notshown). Temperatures may be selected that assist the shift in laser beamfrequencies to a desired region of interrogation. In some embodiments,excitation beam 6 delivered by excitation laser 4 may be modulated witha sine wave at a modulation index that is less than or equal to theDoppler width of the molecular absorption line widths. The term“modulation index” as used herein is defined as the frequency range overwhich a sine wave varies the excitation laser 4.

Spatial Filtering

Laser beam 6 transmitted from laser 4 may be focused in concert withvarious spatial filtering devices including, e.g., focusing lenses 10and 12, slits 14, pin holes 16, distributed feedback (DFB) gratings,including combinations of these various devices. Spatial filteringdevices may be further used to suppress reflections of light back toexcitation laser 4. Eliminating reflected light may eliminate feedbacknoise in the absorption signal. For example, spatial filters including,e.g., slits 14 and pin holes 16 may be positioned at a tilt angle thatselects a subset of rays from excitation beam 6 at a selected pointingangle (defined by an angle phi) and selected beam spread defined by adivergence angle (theta) into the absorption (transmission) device 30.“Pointing angle (phi)” as used herein is an angle representing theangular variation from the central axis of laser beam 6, describedfurther in reference to FIG. 2. “Divergence angle (theta)” as usedherein refers to the angle at which the laser beam diverges or convergesfrom the central axis that defines the spread of the of laser beam 6.“Central axis” as used herein means the optical axis of the laser 4.

In some embodiments, lenses 10 and 14 may be collimating (focusing)lenses that deliver excitation beam 6 from laser 4 such that theexcitation beam 6 is delivered off-axis (i.e., at an angle that isoffset from the central axis of excitation beam 6) into absorption(transmission) device 30.

In some embodiments, lens 10 may be an external aspheric (e.g., F/1)lens constructed of a transmission material such as zinc selenide (ZnSe)that includes a broadband anti-reflective (AR) coating configured totransmit wavelengths from about 0.6 μm to 16 μm suitable for mediumwavelength infra-red (i.e., MWIR) operation.

In some embodiments, excitation beam 6 may be extracted through anoff-axis pin hole 16 to absorption device 30 that suppresses feedbacknoise back to excitation laser 4.

In some embodiments, lens 14 may be a plano-convex, AR-coated silicon(Si) lens (e.g., 50-mm focal length, with >98% bandpass in the MWIR)positioned, e.g., in front of pin hole 16 (e.g., ˜1.5-mm diameter), butlens types are not intended to be limited thereto.

Optical Isolation

Laser 4 may be coupled to an optical isolation device 18 positionedbetween excitation laser 4 and absorption device 30 (discussed furtherin reference to FIGS. 2a-2c herein). “Optical Isolation Device” as usedherein means a device that delivers turbulence 26, e.g., through amedium 24 or turbulence to elements present within the medium 24 as afunction of time. The term “turbulence” as used herein refers to randomspatial and temporal variations introduced in the medium through whichthe laser beam traverses or that is delivered across the propagationpath of the laser beam that alters the index of refraction of themedium. The spatial and temporal variations prevent light from reachingthe excitation laser when reflected from the absorption device reachingback to the excitation laser. Turbulence may be induced in concert withvarious gradients in the medium that introduce variations across thepropagation path of the light beam from the laser en route to theabsorption (transmission) device. The term “gradient” used herein meansa randomly varying distribution of elements (e.g., particulates) presentwithin the medium that introduce variations in the propagation path ofthe light beam. Gradients include, but are not limited to, e.g.,velocity gradients, temperature gradients, heat gradients (i.e.,gradients defined by a random distribution of heat waves), densitygradients, sonic gradients, aerosol gradients, particulate gradients,and/or other gradients.

Medium 24 may be a fluid medium, a gaseous medium, a liquid medium, anaerosol-containing medium, a particulate-containing medium, orcombinations of these various media. Medium 24 may be an open medium anenclosed medium. For example, in some embodiments, medium 24 may becontained within an enclosure 23 or vessel 23. Elements in medium 24 mayinclude, e.g., liquids, gases, particulates, aerosols, heat waves, sonicwaves, and like elements that can define a gradient.

“Optical isolation” as used herein refers to the process whereby theexcitation laser may be isolated from reflected light that returns backto the excitation laser stemming from an optically coupled absorption(or transmission) device. Reflected light introduces feedback noise intothe absorption (transmission) spectrum. Optical isolation in concertwith the present invention may be achieved by inducing random spatial(i.e., 3-D positions in space) and temporal (time) variations in theindex of refraction of a medium positioned between the excitation laserand the absorption (transmission) device. These random variations may beintroduced by a turbulence generator into the medium across thepropagation path through which the laser beam traverses en route to theabsorption device, or a particulate (e.g., aerosol) generator thatdisperses particles of varying size and velocity in the medium throughwhich the light beam from the excitation laser is delivered thatrandomizes the pointing angle and the spread of the light beam as afunction of time while the light beam is en route to the absorption (ortransmission) device. The turbulence or particulates prevent feedbacknoise from appearing in the resulting absorption spectrum (see FIG. 7).

Optical Isolation Device 18 may be configured to scatter light deliveredfrom laser 4 through medium 24 en route to capillary waveguide 30detailed hereafter such that light incident upon, and reflected from,the capillary waveguide 30 experiences scattering conditions and shiftsin the pointing angle and beam spread that are different than conditionspresent for the incident light. The change in pointing and beam spreadmay serve to isolate laser 4 from reflected light, which serves tosuppress optical feedback and resulting feedback noise in the absorptionspectrum.

In particular, turbulence may yield a random spatial and temporalvariation in the index of refraction of medium 24 or a selected gradientin delivery medium 24 that may suppress feedback noise induced byreflection of the excitation beam 6 incident upon, and from within, theabsorption device 30 back to excitation laser 4. Turbulence induced inthe medium may provide a signal-to-noise ratio (SNR) enhancement of atleast about a factor 2 or better.

Optical isolation device 18 may include a turbulence generator 20 orrandomization device that generates turbulence 26 in a medium (open orenclosed) 24 across the propagation path through which the laser beam 6traverses en route to absorption device 30.

Turbulence Generators

Turbulence generators 20 may include, but are not limited to, e.g., heatsources, gas sources, forced gas sources, flowing gas sources, aerosoldevices, nebulizing devices, flame sources, sonic sources, liquidsources, forced liquid sources, flowing liquid sources, includingcombinations of these various devices.

In some embodiments, the turbulence generator may be a heat gunconfigured to deliver a random spread or distribution of heat waves ofvarying temperature across the propagation path of light beam.

In some embodiments, turbulence generator may be coupled to an enclosedliquid through which the light beam is directed that provides thevariation in the index of refraction of the light beam when deliveredfrom laser device to absorption (or transmission) device when directedthrough the liquid.

In some embodiments, the randomization source may be an aerosolgenerator configured to deliver a random spread of aerosols orparticulates dispersed in the medium with a random distribution of sizesand velocities as a function of time across the propagation path of thelight beam that is delivered through the volume of aerosols to theabsorption (or transmission) device that induces random spatial andtemporal variations in the scatter of the beam as it passes through theaerosols or particulates in the medium while en route to the absorption(transmission) device. In some embodiments, the turbulence generator maybe a vapor-nebulizing device. In some embodiments, the randomizationsource may be a sonic device configured to deliver a random spread ofsonic waves at a plurality of temperatures above ambient across thepropagation path of the light beam from the laser that provides therandom spatial and temporal variation in the medium when the light beamis delivered through the spread of sonic waves to the absorption deviceand/or the transmission device.

Absorption Device

The LA-CAS spectrometer 100 may also include an absorption (or atransmission) device 30 such as a hollow waveguide.

In some embodiments, absorption device 30 may be a capillary waveguideconfigured to receive a sample gas for analysis containing analytes thatabsorb (or transmit) in the infra-red wavelength region when anexcitation beam from an excitation laser is introduced into the samplegas therein.

In some embodiments, the waveguide may have an internal diameter (I.D.)that is 1-mm and a length that is 0.8 meters. However, dimensions arenot intended to be limited. In some embodiments, the waveguide may havean internal diameter that is constant. In some embodiments, thewaveguide may be of a tapered design, with an internal diameter thatvaries as a function of length (i.e., one end has a smaller diameterthan the other end) to suppress noise from reflections within thecapillary.

In some embodiments, gas volumes in the absorption (transmission) devicemay below about 0.6 cm³. In other embodiments, gas volumes may begreater than about 0.6 cm³. No limitations are intended.

In some embodiments, waveguide 30 may include coatings optimized forselected laser wavelengths.

In some embodiments, waveguide 30 may include Louisville optics, e.g.,as detailed by Harper et al. in Laser-based Spectroscopic DetectionTechniques, U.S. Pat. No. 7,102,751, which reference is incorporatedherein in its entirety.

Capillary waveguide 30 can allow analyses of samples at total gaspressures below about 25 Torr (0.03 atm), more preferably below about 2Torr (0.003 atm), and yet more preferably between about 1 Torr (0.001atm) and 2 Torr (0.003 atm).

Absorption device 30 may also include transmission windows 32 and 38positioned at respective ends of absorption device 30 that deliverselected IR wavelengths that are absorbed by analytes in absorptiondevice 30 that provide for analysis of these analytes.

In various embodiments, the waveguide may be a capillary waveguide thatincludes IR transmission windows composed of, e.g., ZnS that transmitwavelengths from about 0.6 μm to 16 μm. In some embodiments, thetransmission windows may be composed of sapphire that transmitswavelengths in the range from about 0.15 μm to 5 μm. In otherembodiments, transmission windows may be made of other transmissionmaterials known in the art may be that transmit IR wavelengths withinselected and suitable ranges for absorption by analytes in thewaveguide. All transmission materials as will be selected by those ofordinary skill in the art in view of the disclosure are within the scopeof the invention. No limitations are intended.

Absorption device 30 may be coupled to low-dead-volume or vacuum tees 34and 40 and vacuum valves 36 and 42 that allow sample gases and analytesto be introduced into waveguide 30 and exchanged from waveguide 30 forabsorption measurements therein.

In some embodiments, a gas inlet 48 may be employed to provide gastransfer rates into absorption device 30 at low inlet pressures, e.g.,between about 0.5 Torr (7×10⁻⁴ atm) and about 20 Torr (0.03 atm). Inlet48 may be of a small dead volume design to provide gas-tight operationinto waveguide 30. Gas inlet 48 may couple to any of a variety ofinstrument systems including, but not limited to, e.g., gaschromatographs (GC), laser-ablation and catalytic reactor devicesdetailed further herein, and other analytical devices. No limitationsare intended.

In some embodiments, waveguide 30 may be operated in a static mode. Insome embodiments, waveguide 30 may be operated in a continuous (dynamic)mode.

For example, in some embodiments, outlet valve 36 may couple to a vacuumsystem (not shown) allowing exchange of static samples or a controlledcontinuous flow of a sample in a dynamic or a continuous sampling modeat selected and selected and/or suitable vacuum pumping speeds and gastransfer rates. Position of valves in the present invention is notintended to be limited to exemplary embodiments described herein.

In some embodiments, waveguide 30 may be coupled to a laser ablationdevice and a catalytic reactor as detailed hereafter, which permitsconversion of solid samples into molecular gases suitable for absorptionmeasurements within waveguide 30.

Laser Ablation and Catalytic Combustion Conversion

Spectrometer (LA-CAS) 100 may include a laser ablation device 44 coupledwith a catalytic (or combustion) reactor 46 that provides ablation ofsolid samples and conversion of the ablated particulates formingmolecular gases. The molecular gases may be introduced to absorptiondevice 30 for analysis in spectrometer (LA-CAS) 100.

Detector

The LA-CAS spectrometer (LA-CAS) 100 may also include a detector 52 thatis coupled to absorption device 30 and to a detection device 54configured to detect absorption features for one or more analytes in asample gas when introduced into absorption device 30. The detectortransmits absorption (transmission) signals. Detectors suitable for usemay include infra-red sensitive detectors such as indium-antimonide(InSb) detectors configured to produce an output current that isproportional to the intensity of the laser light transmitted from theabsorption device that is now incident on the detector resulting fromdetection of absorption (transmission) signals generated in thewaveguide during analysis.

Turbulence Generator Operation

FIG. 2a shows a turbulence generator 20 coupled to a medium 24containing a gas dispersed in an open volume. In the figure, turbulencegenerator 20 induces turbulence in the gas 24 by delivering randomthermal and velocity gradients across the propagation path of excitationbeam 6 delivered through the gas in the open medium 24. In someapplications, turbulence may be induced in the gas by delivering heatedand turbulent air from the generator. In the figure, excitation beam 6may be delivered through medium 24 to absorption device 30. Whendelivered through the turbulent medium, laser beam 6 may be spread froma parallel delivery path by a distance defined by a divergence angle(theta) 60. Any reflected light 56 from absorption device 30 must passthrough medium 24 again. The random spatial and temporal variations inthe index of refraction of the medium 24 due to the induced turbulenceensures that laser beam divergence angle (theta) 60 and laser beam 6pointing angle (phi) 58 are different for reflected light 56, whichprevents reflected light 56 from being incident on laser 4 and preventsfeedback-induced noise in the absorption spectrum.

FIG. 2b shows a turbulence generator 20 (e.g., a sonic generator or aheat generator) coupled to an enclosed volume 24 containing, e.g., anenclosed fluid or an enclosed gas. Again, turbulence generator 20induces turbulence in the fluid or gas contained within the enclosedvolume 24 that delivers, e.g., random sonic and/or thermal agitation inthe enclosed medium 24. In the figure, excitation beam 6 may bedelivered through medium 24 to absorption device 30. When deliveredthrough the turbulent medium 24, laser beam 6 may be spread from aparallel delivery path by a distance defined by a divergence angle(theta) 60. Any reflected light 56 from absorption device 30 must passthrough medium 24 again. Random spatial and temporal variations in theindex of refraction of medium 24 containing the gas due to the inducedturbulence ensures that laser beam 6 includes a divergence angle (theta)60 and a pointing angle (phi) 58 that are different for the reflectedlight 56, which prevents reflected light 56 from being incident on laser4 and prevents feedback-induced noise in the absorption spectrum.

FIG. 2c shows a generator 22 of an aerosol or particulate deliverydesign used as a component of an optical isolation device 18. In thefigure, generator 22 is configured to deliver a random spread ofaerosols or particulates with a random distribution of sizes andvelocities across the propagation path of the light beam 6 when thelight beam 6 is delivered to the absorption (or transmission) device 30.For example, when the light beam 6 is directed through the spread ofaerosols or particulates in the medium 24, the pointing angle beam 6 andthe spread of the light beam as a function of time are randomized. Inparticular, when the laser beam 6 is delivered through the aerosols orparticulates in the medium, any reflected light 56 from absorptiondevice 30 must pass through the medium 24 again. The random distributionof aerosols of various sizes and velocities in medium 24 ensures thatthe divergence angle (theta) 60 and the pointing angle (phi) 58 of laserbeam 6 are different for the reflected light 56, which preventsreflected light 56 from returning back to the laser 4, therebypreventing feedback-induced noise in the absorption spectrum.

Conversion of Solid Samples

FIG. 3 shows exemplary devices that may be coupled to CAS 100 to convertsolid samples into molecular gases suitable for analysis by the presentinvention. As shown in the figure, CAS 100 may further include a laserablation device 44 that couples to a catalytic reactor 46. Laserablation device 44 may ablate the sample (including, e.g., biologicalmaterials) and form sample particulates, e.g., as detailed by Moran etal. (Rapid Commun. Mass Spectrom. 2011, 25, 1282-1290), which referenceis incorporated herein in its entirety. Ablated sample particulates fromablation device 44 may be delivered through a catalyst in catalyticreactor 46, which converts the particulates into a molecular gascontaining isotopologues representative of the ablated sample. Moleculargases formed in reactor 46 may be subsequently transferred via atransfer device 50 (described further herein in reference to FIG. 4 andFIG. 5) into the wave guide 30 for determination therein. Sampleparticulates may be combusted forming gases that may be analyzed atpressures below 25 Torr (0.03 atm). Molecular gases stemming from theconverted and combusted sample may be transferred through a transferdevice detailed hereafter to the capillary absorption device 30(described previously in reference to FIG. 1) for analysis.

Cryogenic Transfer Devices

FIG. 4a shows one embodiment of a transfer device 150 of a cryogenicdesign configured to transfer (deliver) analytes in a molecular gas froma catalytic reactor 46 at a first (e.g., high) pressure and deliver theanalytes in the molecular gas into absorption device 30 at a second(lower) pressure without loss of analytes. Transfer device 150 mayinclude a cryogenic trap 63 with a trapping volume 68 that isconfigured: 1) to receive the molecular gas delivered from the catalyticreactor 46, e.g., through a first valve 64 at a first (high) pressure.Trapping volume 68 of cryogenic trap 63 may be coupled, e.g., to acondensing (freezing)/evaporation device 70 that condenses and traps theanalyte-containing molecular gas. For example, in the cryo-trappingphase, valve (V1) 64 may be opened to receive a flow of gas fromcatalytic reactor 46 into trapping volume 68 of cryogenic trap 63 at atemperature below the freezing point of the molecular gas such thesample gas is condensed, frozen, and trapped as a solid therein whileallowing a carrier gas such as helium (He) with a higher freezingtemperature to flow through the trap 63. A purge and/or carrier gas 62such as helium (He) may be introduced into cryogenic trap 63, e.g.,through a second valve (V2) 66 to assist with transfer of sample gasesfrom trapping volume 68 to capillary absorption spectrometer 30. Valve(V1) 64 may be closed and trapping volume 68 containing thecryogenically trapped sample may be evacuated (e.g., through a vacuumsystem valve of the LA-CAS described in reference to FIG. 1) to removeexcess carrier gas that yields a lower pressure, e.g., between about 1Torr (0.001 atm) and about 25 Torr (0.03 atm) suitable for introductionof the molecular gas into capillary absorption device 30 for analysistherein. In this manner, analytes in cryogenic trap 63 may be isolatedto give gas pressures in trap 63 that match the second (lower) pressureof absorption device 30. Following evacuation, valve 36 to vacuum systemmay be closed and freezing/evaporation device 70 may heat thecryotrapped gas in trapping volume 68 above the evaporation temperatureof the molecular gas, allowing it to fill absorption spectrometer 30 atthe second (lower) pressure. Pressures suitable for use in the transferdevice are not intended to be limited. However, pressures may include afirst (high) pressure of from about 760 Torr (1 atm) down to 100 Torr(0.13 atm). Second pressure may be a pressure below about 25 Torr (0.03atm). In the instant configuration, cyrotrapping volume 68 of cryogenictrap 63 is separate and distinct from the capillary absorptionspectrometer 30 volume.

FIG. 4b shows another embodiment of a cryogenic transfer device 250 ofthe present invention. In this embodiment, freezing/evaporation device70 is positioned in thermal contact with a selected section or portionof capillary waveguide 30 such that the trapping volume 68 of cryogenictrap 63 is positioned within, and at the leading end of, capillarywaveguide 30 to provide cryotrapping of the molecular gas in thatsection or portion of capillary waveguide 30. In the present embodiment,the capillary waveguide 30 functions both as the transfer device at thefirst high pressure and the capillary waveguide 30 at the second lowerpressure. The present embodiment eliminates potential for introducingdead volumes during the evaporation phase immediately prior to analysiswhich can reduce molar sensitivity or diluting the cryotrapped samplethat can result from addition of an extra volume external to capillarywaveguide 30. All other components and operation are as detailed abovefor FIG. 4 a.

Conduction-Limited Transfer Devices

FIG. 5a shows yet another embodiment of a transfer device 350 of aconduction-limited design configured to deliver analytes in a moleculargas from a catalytic reactor 46 at a first (e.g., high) pressure anddeliver the molecular gas containing the analytes into absorption device30 at a second (lower) pressure without a loss of analytes. Pressuressuitable for use are not intended to be limited, as detailedhereinabove. Transfer device may include a pin hole (orifice) 72positioned to impose a conductance barrier for transfer of moleculargases between two different pressures, e.g., a first high pressure and asecond lower pressure. Dimensions of pin hole 72 are not limited, andmay be selected such that molecular gases are transferred through pinhole 72 at a selected rate and at a selected pressure into absorptiondevice 30. For example, transfer of the gas through the pin hole 72reduces pressure of the gas from the first high pressure to the 2^(nd)lower pressure such that it is compatible with the inlet pressure intoabsorption device 30 at the lower pressure.

FIG. 5b shows yet another embodiment of a conduction-limited transferdevice 450 that operates under a graded pressure reduction regime,providing continuous transfer of analytes from the catalytic reactor 46to the absorption device 30. In the figure, transfer device 450 has theform of a capillary tube 74 positioned to impose a conductance barrierfor transfer of molecular gases between two different pressures, e.g., afirst high pressure and a second lower pressure. Length and internaldiameter dimensions of capillary tube 74 are not limited, and may beselected such that analytes in the molecular gas received from catalyticreactor 46 may be transferred into capillary tube 74 at a selected rateand at a first high pressure, and then undergo a selected and gradedpressure reduction, and then to exit capillary tube 74 at a second(lower) pressure compatible with introduction into IR absorption device30. Analytes may be continuously transferred at selected rates fromcatalytic reactor 46 to IR absorption device 30 for analysis.

Modulation Circuit

FIG. 6 shows a schematic of a (signal) modulation device circuit 2 ofLA-CAS 100. Modulation device 2 may be configured to modulate thewavelength of excitation beam 6 delivered by excitation laser 4, whichmodulations may be delivered as outputs to excitation laser 4.Modulation device 2 may include a linear current ramp source 122 [e.g.,a Digital Signal Oscillator (DSO)] that provides a repetitive linearramp current to a DC power (offset) (summing) device 124. The DC offsetdevice 124 provides a DC offset current. The linear current from thelinear current source 122 determines the wavelength range over which thelaser frequency is scanned. The value of the DC offset currentdetermines the starting value of the laser frequency in the frequencyscan. The maximum value of the current from the linear current rampsource 122 determines the ending value of the laser frequency scan. Theslope of the current from the linear ramp device 122 may determine therate at which the laser frequency is scanned. The DC offset device 124then sums the linear ramp current obtained from the linear currentsource 122 together with the DC offset current from the DC offset device124, e.g., as detailed by Taubman et al. in U.S. Pat. No. 7,957,441,which reference is incorporated in its entirety herein.

Modulation device 2 may further include a dither-modulated currentsource 128 [e.g., a DSO] that delivers a dither-modulated current (e.g.,AC delivered at 1f) as an output in which the frequency of the modulatedcurrent is ½ the in-quadrature condition of a residual amplitudemodulation distortion frequency (response) of excitation laser 4. Theterm “in-quadrature” as used herein means the frequency oscillations(modulations) in the excitation beam differ or are separated in phase by90° (π/2 radians or λ/4) from the amplitude oscillations (ormodulations). In some embodiments, dither-modulated current source 128may be operated at frequency (f) that includes a crossover frequencynear 95 kHz, e.g., as detailed by Kelly et al. [Review of ScientificInstruments, 83, 023101 (2012)]. No limitations are intended.

Driving Circuit

The output (AC) from the dither-modulated current source [DSO F] 128 maybe combined with the repetitive linear ramped current delivered from thelinear current ramp source [DSO] 122 along with the DC offset currentfrom the DC offset source 124 and provided as an output to a 2^(nd)order or higher Bias-Tee (diplexer) 126 positioned in modulation device2. Bias-Tee (diplexer) 126 may deliver a combined, summed, and thustotal modulated current signal to excitation laser 4 as a function oftime. Bias-tee 126 may also suppress harmonics from the repetitivelinear current ramp source 122 and sub-harmonics from thedither-modulated current source 128 such that the amplitude of theharmonics and sub-harmonics is at least 60 dB lower than the totalmodulated current waveform selected and provided to excitation laser 4.The term “harmonic” means an integer multiple of a fundamentaloscillation frequency delivered by a signal oscillator that may causeundesired noise in a detected signal. The term “sub-harmonic” means afraction of the fundamental oscillation frequency delivered by a signaloscillator that may cause undesired noise in a detected signal.

Phase-Sensitive Detection Device Circuit

FIG. 6 further shows a phase-sensitive detection device circuit 54 thatcouples with modulation device circuit 2. Phase-sensitive detectioncircuit 54 suppresses noise in the absorption signal arising from theRAM distortion frequency received from excitation laser 4 and fromharmonic distortion frequencies received from dither-modulated currentsource 128. Phase-sensitive detection circuit 54 may include a firstcircuit segment (leg) 102 having a diplexer/band-pass filter 112 of a2^(nd) or greater order that delivers a detection signal 106 fromabsorption device 30 as an input to a double-balanced mixing device 114.First circuit segment 102 may also include a bias-tee 108 of a 2^(nd) orgreater order configured to receive the detection (absorption) signal106 from the detector (FIG. 1). Detection signal 106 may be amplified,e.g., with a pre-amp device 110 prior to being introduced todiplexer/bandpass filter 112 that filters out amplification noise fromdetection signal 106 before it is introduced to double mixing device114.

Phase-sensitive detection circuit 54 may also include a second circuitsegment (leg) 104 that includes a Band-Pass Filter (BPF) 118 configuredto deliver a reference (drive) signal free of harmonic andintermodulation distortion as a separate input to double-balanced mixingdevice 114.

In some embodiments, the reference drive signal delivered to thebandpass filter 118 may be at a frequency (i.e., 1f) provided by thedither-modulated current source 128 that is ½ the frequency for thein-quadrature condition of the residual amplitude modulation distortionfrequency (response) of excitation laser 4. The term “1f” refers to thedrive frequency of the dither-modulated current source 128. Signalrecovery can be achieved at (1f) when the amplitude of the laser deviceis constant over the wavelength range being scanned. Thus, nolimitations are intended.

In some embodiments, a 2^(nd) DSO device 120 may be inserted betweendevice [DSO F] 128 and bandpass filter 118 to deliver a reference ordrive (2f) signal [e.g., for (2f) signal recovery] to bandpass filter128 at a frequency (i.e., 2f) that is 2 times the frequency provided bythe dither-modulated current source [DSO F] 128. The 2f drive frequencyprovided by DSO 120 device may be synchronized such that it is in-phasewith the output (1f) frequency delivered from dither-modulated currentsource 128. The term “2f” (or second harmonic detection or WaveformModulated detection) refers to a drive frequency or a detectionfrequency that is twice the magnitude of the reference frequencydelivered from the dither-modulated current source 128. A (2f) frequencymay be used when the amplitude of the intensity of the laser beam is notconstant over the wavelength range being scanned in order to eliminateslope in the baseline, delivering a flat baseline in the detectionsignal.

Double-balanced mixing device 114 may then deliver an output signal madeup of various combinations of signals (i.e., mixed signals) receivedfrom the respective inputs. For example, double-balanced mixing device(DBM) 114 may couple to a 2^(nd) or greater order diplexer (bias-T) 122configured to: 1) receive the mixed output signal from the DBM 114 as aninput and 2) to isolate a selected absorption response signal (seediscussion FIG. 7) for one or more selected analytes present in themixed signal. The 2^(nd)-order or greater Diplexer 122 may be configuredto receive an output signal from the double balanced mixing device 114as an input and to deliver a distortion free response signal as anoutput at a selected frequency therefrom. In various embodiments, the2^(nd)-order or greater diplexer 122 may deliver the absorption responsesignal isolated from 1-times (1f) or 2-times (2f) the dither-modulatedfrequency delivered from the dither-modulation device 128 that is also90 degrees out-of-phase with the RAM distortion frequency provided toexcitation laser 4. In some embodiments, the 2^(nd)-order or greaterDiplexer may deliver a response signal with a frequency selected in therange from about 100 Hz to about 30 KHz with an isolation of about 30 dBper decade at the upper cutoff frequency. The isolated response signalmay be observed in concert with a CPU 130 or other transient signalrecording device.

FIG. 7 compares (2f) absorption signals for representative isotopologuesof carbon dioxide containing ¹³C and ¹²C, respectively, recovered withand without use of the optical isolation device of the presentinvention. In the figure, signals were obtained at twice the frequencyof the dither-modulated current source. Results show the reduction infeedback noise produced by the incident laser beam on the capillary waveguide stemming from reflection from the absorption device back to theexcitation laser. Results collected with the optical isolation device(FIG. 1) of the present invention shows that feedback noise is reducedby a factor of at least about 50 compared with results collected absentthe optical isolation device. However, reduction factors are notintended to be limited. The reduction factor may be calculated as theratio of the total integrated intensity of the absorption features tothe standard deviation (SD) of variations (noise) observed in thebaseline.

Detection Limits

TABLE 1 compares instrumental gas-phase detection limits (DL) of LA-CAS100 to commercial infrared absorption-based isotopic analyzers usinginternationally certified isotopic CO₂ gas standards.

TABLE 1 Compares molar detection limits and isotopic precisions (‰) atgiven analysis (dwell) times for isotopic measurements of CO₂ ofcommercial isotopic analyzers compared with the LA-CAS. EffectiveMeasured Detection Precision, Limit ‰ Total CO₂ Molar and VolumePressure Concentration Sensitivity (Dwell System (mL) (Torr) (ppm)(Moles) Time) 1^(a) 30 300 300 1.6 × 10⁻⁷  0.5 (100 sec)  2^(b) 120 300300 6.4 ×10⁻⁷  0.25 (60 sec) 3^(c) 300 25 300 1.3 × 10⁻⁷  0.2  (1 sec)LA-CAS 0.63 4.0 749 1.1 × 10⁻¹⁰ 0.6 (10 sec) LA-CAS 0.63 2.5 4500 4.2 ×10⁻¹⁰ 0.1 (10 sec) LA-CAS 0.63 2.0 390 2.0 × 10⁻¹⁰ 2 (10 sec)^(a)Picarro CRDS instrument, E. H. Wahl et al. in “Isotopes Environ.Health Stud.” 42, 21 (2006). ^(b)Los-Gatos ICOS instrument[http://www.lgrinc.com]. ^(c)Aerodyne 7.3-meter Herriott cell [see J. B.McManus et al. in “Isotopes Environ. Health Stud.”, 46, 49 (2010); andD. D. Nelson et al. in “Appl. Phys. B”, 90, 301 (2008).

In TABLE 1, isotopic precision (‰) is stated in standard delta (δ)notation used in isotopic measurements, in which stable isotopiccompositions of low-mass (light) elements such as oxygen, hydrogen,carbon, nitrogen, and sulfur are normally reported as “delta” (δ) valuesin parts per thousand (denoted as ‰) enrichments or depletions relativeto a standard of known composition. The symbol (‰) is spelled out inseveral different ways: per mil, per mil, per mill, or per mille. Theterm “per mill” is the ISO term, but is not yet widely used. Delta (δ)values may be calculated, as given by Equation[1] below:

(‰)=(R _(sample) /R _(standard)−1)1000  [1]

Here, “R” is the ratio of the heavy to light isotope in the sample orstandard. For the elements sulfur, carbon, nitrogen, and oxygen, theaverage terrestrial abundance ratio of the heavy to the light isotoperanges from 1:22 (sulfur) to 1:500 (oxygen); the ratio ²H:¹H is 1:6410.A positive (δ) value means that the sample contains more of the heavyisotope than the standard. A negative (δ) value means that the samplecontains less of the heavy isotope than the standard. As an example, aδ¹⁵N value of +30‰ means that there are 30 parts-per-thousand (or 3%)more ¹⁵N isotopes in the sample compared with the standard.

As shown in TABLE 1, LA-CAS 100 of the present invention provides asensitivity exceeding other infrared absorption detection methods.LA-CAS 100 with 1-mm ID hollow waveguides (HWGs) of nominal 1-m pathlength can resolve δ¹³C variances to less than 1‰ with total samplequantities down to 100 picomoles (pmols) using commercial standards. TheLA-CAS has also demonstrated molar sensitivities down to 2 picomoles at1‰, with projected improvements down to 100 femtomoles.

APPLICATIONS

The LA-CAS of the present invention enables spatially resolved isotopicimaging in a variety of sample types for fundamental biological andchemical research and forensic applications, including, e.g., thinfilms, polymers, forensic samples including, e.g., hair (both human andanimal), tree growth rings, and microbial systems. And, LA-CAS can beperformed on samples under ambient atmospheric conditions that requireno preparation beyond mounting the sample. Stable isotope analysespermit tracking of physical, chemical, and biological reactions invarious sample materials in physical, biological, and chemicalprocesses, interactions, and mechanisms at various spatial scalesranging from the atmosphere to individual microorganism cells. TheLA-CAS may also be configured to study isotopic ratios of CO₂ and othergases produced by catalytic combustion of evolved particulates createdby UV laser ablation of nonvolatile organics, and can be used to studyCO₂ and other molecular gases directly from various nichebio-compartments including waste gases generated from soil bacteria andother environments.

The LA-CS also provides highly sensitivity isotope ratio measurements onextremely small samples with volumes less than or equal to about ˜0.5mL.

The present invention also eliminates optical feedback in continuous(CW) Quantum Cascade (QC) lasers.

While exemplary embodiments of the present invention have been shown anddescribed, it will be apparent to those skilled in the art that manychanges and modifications may be made without departing from theinvention in its true scope and broader aspects. The appended claims aretherefore intended to cover all such changes and modifications as fallwithin the spirit and scope of the invention.

What is claimed is:
 1. A capillary absorption spectrometer for analysisof low-volume samples, comprising: an excitation laser configured tosupply a non-pulsed excitation beam to an absorption device having anabsorption volume configured to receive the excitation beam from theexcitation laser into a sample gas introduced therein; a modulationdevice comprising a second or higher order bias-T diplexer coupled tothe excitation laser that suppresses harmonics from a repetitive linearcurrent ramp source therein and sub-harmonics from a dither-modulatedcurrent source therein such that the amplitude of the harmonics and thesub-harmonics is at least 60 dB lower than the modulation waveformselected and provided to the excitation laser; and an optical isolationdevice disposed between the excitation laser and an absorption devicethat randomizes the pointing angle and the spread of the excitation beamthat suppresses feedback noise induced by reflection of the excitationbeam incident upon, and from within, the absorption device back to theexcitation laser.
 2. The spectrometer of claim 1, further including alaser ablation device that couples to a catalytic reactor and a transferdevice, the laser ablation device is configured to deliver ablatedsample particulates to the catalytic reactor for combustion andconversion of the sample particulates into a molecular gas, the transferdevice is configured to transfer the molecular gas containing analytesfrom the catalytic reactor into the absorption device for analysistherein.
 3. The spectrometer of claim 2, wherein the transfer devicetransfers analytes from the catalytic reactor at a first (high) pressureand delivers the analytes into the absorption device at a 2^(nd) (lower)pressure without a loss of analytes.
 4. The spectrometer of claim 2,wherein the transfer device is a conduction-limited transfer device thatprovides continuous transfer of analytes from the catalytic reactor atthe first (high) pressure to the IR absorption device at the second(lower) pressure in concert with a graded reduction in pressure.
 5. Thespectrometer of claim 2, wherein the transfer device is a cryogenictransfer device that includes a cryogenic trap.
 6. The spectrometer ofclaim 5, wherein the cryogenic trap is configured to receive analytesfrom the catalytic reactor at the first (high) pressure and tosimultaneously condense and trap the analytes therein.
 7. Thespectrometer of claim 5, wherein the cryogenic trap is configured toisolate and evacuate the transfer device to match the second (lower)pressure of the absorption device.
 8. The spectrometer of claim 5,wherein the cryogenic trap is configured to evaporate and transfer theanalytes into the absorption device at the second (lower) pressure. 9.The spectrometer of claim 1, wherein the excitation laser is acontinuous-wave laser.
 10. The spectrometer of claim 1, furtherincluding one or more spatial selection (slit) devices disposed todeliver the excitation beam in an off-axis direction through the mediumof the optical isolation device to the absorption device.
 11. Thespectrometer of claim 1, wherein the absorption device is a capillaryoptical waveguide.
 12. The spectrometer of claim 1, wherein theabsorption device is a tapered hollow waveguide with an internaldiameter that varies as a function of length, or an optical fiber with afixed internal diameter.
 13. The spectrometer of claim 1, wherein theabsorption device defines a volume configured to contain a total gaspressure at or below 25 Torr (0.03 atm).
 14. The spectrometer of claim1, wherein the modulation device includes a dither-modulated currentsource that delivers a dither-modulated frequency that is tuned to afrequency that is one-half (½) the Residual Amplitude Modulation (RAM)distortion frequency (response) of the excitation laser.
 15. Thespectrometer of claim 1, wherein the modulation device includes arepetitive linear current ramp source and a dither modulated currentsource each configured to deliver frequencies that are non-integerfractions of fundamental reference frequencies of these sources thatsuppresses cross talk arising from phase and/or frequency differencesbetween these respective sources.
 16. The spectrometer of claim 15,wherein the repetitive linear current ramp source delivers a frequencythat is at least a factor of 5 below the frequency delivered by thedither-modulated current source.
 17. The spectrometer of claim 1,further including a detection device operatively coupled to theabsorption device configured to detect absorption features for one ormore analytes when present in the sample gas in the absorption device.18. The spectrometer of claim 17, wherein the detection device includesa phase-sensitive detection circuit, comprising: a first circuit segmentcomprising a diplexer configured to deliver a detection signal from theabsorption device as an input to a double-balanced mixing device; and asecond circuit segment comprising a Band-Pass Filter (BPF) configured todeliver a reference (drive) signal free of harmonic and intermodulationdistortion from the dither-modulated current source of the modulationdevice as a separate input to the double-balanced mixing device.
 19. Thespectrometer of claim 18, wherein the double-balanced mixing devicedelivers an output signal comprising combinations of the signalsreceived from the respective inputs.
 20. The spectrometer of claim 18,wherein the double-balanced mixing device (DBM) couples to a diplexer ofa 2^(nd) or greater order configured to receive the mixed output signalfrom the DBM as an input and isolate a selected absorption responsesignal for one or more selected analytes present therein.
 21. Thespectrometer of claim 20, wherein the 2^(nd)-order Diplexer delivers theisolated response signal at 1-times (1f) or 2-times (2f) thedither-modulated frequency of the modulation device that is also 90degrees out-of-phase with the RAM distortion frequency of the excitationlaser.